Thrombomodulin is a key regulator of blood clotting. Oxidation of methionine 388 in thrombomodulin greatly slows the rate at which the thrombomodulin-thrombin complex activates protein C, which, upon activation, slows clot formation. The hypothesis that will be tested is that oxidation of this methionine is elevated in smokers relative to non-smokers. The ultimate aim is to establish if this is an important molecular cause of cardiovascular disease in smokers. Tobacco smoke is known to impose an oxidative stress on the body. The leading cause of premature death in smokers is cardiovascular disease due to smoking. The work will begin with development of one or more immunoassays that are specific for either the reduced or oxidized methionine 388 forms of thrombomodulin. Antibodies will be raised against peptides and recombinant proteins containing the oxidized and reduced forms of this methionine. The accuracy of the immunoassays developed using these antibodies will be confirmed by direct chemical analysis of oxidized and reduced proteins. In this analysis, the purified protein will be digested with proteases. The resulting peptide mixture will then be subjected to chromatography to separate the oxidized and reduced peptides containing the methionine of interest. After validation, these tools will be used to conduct a small scale study of the differences in thrombomodulin 388 oxidation in smokers and non-smokers. The immunoassays will be used to compare the ratio of reduced and oxidized methionine 388 in thrombomodulin found in the urine to the ratio in protein found in the plasma and this information will be correlated with other factors important to blood coagulation. Variation of thrombomodulin oxidation over time will also be followed in a small group of volunteers.